Cooling effects and association of penetrant, non-penetrant cryoprotectors and ascorbic acid on the quality of vitrified bovine preantral follicles

The present study aimed to evaluate the cooling effects and association of penetrant, non-penetrant cryoprotectors and ascorbic acid on the quality of vitrified bovine preantral follicles (FOPAs). Two experiments were conducted. In experiment I, the morphology of FOPAs after cooling followed by vitrification was studied. Ovaries were collected (n=10) from five crossbred heifers aged of 14 to 16 months. At the laboratory 22 ovarian fragments were taken from the cortical region and distributed as followed: two fragments for fresh control (zero hour) submitted to histological analysis and 20 fragments cooled at 4 ºC for four and 24 hours in TCM-199+HEPES+Antibiotics medium. From the 20 fragments cooled, four were fixed as control 4 and 24 hours (two piece each) and the remaining 16 fragments were distributed in four vitrification treatments for each cooling time, respectively: Treatments V4a and V24a: TCM-199 + dimethyl sulfoxide (DMSO) 1.5M + Ethylene glycol (EG) 1.5M; Treatments V4b and V24b: TCM-199 + DMSO 1.5M + EG 1.5M + sucrose (SUC) 0.5M; Treatments V4c and V24c: TCM-199 + DMSO 1.5M + EG 1.5M + Ascorbic Acid (AA) 0.1 mM/L and treatments V4d and V24d: TCM-199 + DMSO 1.5M + EG 1.5M + SUC 0.5M + AA 0.1mM/L. After being vitrified, fragments were stored in liquid nitrogen for three days. Fragments were then heated in solution of decreasing SUC and fixed for histology. In experiment II, the viability of FOPAs after cooling followed by vitrification by Trypan Blue staining (AT) was studied. Bovine ovaries (n = 10) were collected, fragmented and distributed as described in the first experiment, subsequently submitted to mechanical follicular isolation for analysis of viability. The morphology variable was evaluated by SNK test and viability was analyzed by Chi-square Test at 5% probability or the Fisher Exact Test when the number of repetitions was less than 30 follicles. There was no difference in percentage of morphologically normal follicles between fresh control group and control group cooled for 4 h (99.3% and 96.0% respectively; P>0.05), nevertheless, there was reduction of FOPAs morphological integrity of control cooled for 24 h (86%; P0,05). Entretanto, houve redução da integridade morfológica dos FOPAs após 24 h de resfriamento (controle 24 h: 86%; P 0.05). Este mesmo tratamento (V4c) mostrou maior capacidade de preservação em relação aos demais tratamentos de vitrificação (P